Peptide increasing fusiogenic capacity of a gamete

ABSTRACT

The invention relates to a cyclic peptide increasing the fusiogenic capacity of the oocyte arising from the disintegrin loop of fertilin beta, to a pharmaceutical composition comprising said cyclic peptide and to the use thereof, particularly in order to supplement culture media used to carry out in vitro fertilization.

This is a divisional of U.S. application Ser. No. 10/579,921 (publishedas US 2007-0082839 A1), filed May 19, 2006 (issued as U.S. Pat. No.8,883,742), which is a U.S. national phase of international applicationPCT/FR2004/002956, filed 19 Nov. 2004, which designated the U.S. andclaims priority of FR 0313545, filed 19 Nov. 2003, the entire contentsof each of which are hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to cyclic peptides increasing the fusiogeniccapacity of the oocyte and/or the spermatozoon, to pharmaceuticalcompositions comprising the same and to the uses thereof, particularlyin order to supplement culture media used to carry out in vitrofertilization. The inventive peptides typically comprise a domainarising from the disintegrin loop of fertilin beta.

STATE OF THE ART

Fertilization is a complex process leading to the fusion of gametes(spermatozoon and oocyte) to form an embryo. This natural process takesplace in the tubalar ampulla and results in pregnancy after migration ofthe embryo to the uterine cavity.

When a couple is infertile, Medically Assisted Reproduction techniquesare used to obtain embryos in vitro. The embryos are then transferredthrough the vagina into the uterus where they become implanted.

In vitro fertilization simply consists of contacting oocytes andspermatozoa, in a suitable culture medium and in suitable conditions ofpH and temperature, so that fertilization can take place. Methods havebeen widely developed over the past 25 years (Bavister 2002).

When, for reasons that are not always clear, fertilization fails,biologists can resort to assisted fertilization techniques whereby asperm cell is injected directly into the oocyte cytoplasm. However, thedevelopment of microinjected embryos is not as good as that observedafter spontaneous fertilization. In fact, the pregnancy rate aftertransfer of such embryos is lower than after simple in vitrofertilization (25.4% vs 26.5%, on over 20,000 attempts) (FIVNAT, 2001).Likewise, embryos produced by microinjection are less tolerant tofreezing than those obtained by IVF (BLEFCO 2001; Simon et al., 1998).For instance, embryo lysis is more likely during thawing and a lowerimplantation rate is obtained.

Lastly, it is known that activation of the oocyte followingfertilization is mediated by calcium oscillations induced by asperm-derived factor (Swann 1999). These oscillations have a regulatedamplitude and frequency on which the quality of the embryo depends(Swami 1999). Short-circuiting the membrane steps of the gameteinteraction modifies the nature of these calcium waves for two reasons.The physiological interaction between the membranes does not take place,and the trauma to the cell from introducing the microinjection pipetteinto the cytoplasm causes calcium outflow from the reservoirs (Tésariket al. 2002). While this outflow ensures oocyte activation, it can alsoperturb the subsequent development of the embryo.

The study of the process of fusion between the sperm and egg membranesat fertilization has partially revealed the mechanism of membranefusion.

Formation of a membrane molecular complex takes place at the oocytesurface. Said complex is induced by the spermatozoon at fertilization.Its composition and mechanism of action have not been fully elucidated,but it is known that inhibition of its formation leads to inhibition ofgamete fusion. Fertilization is therefore clearly related to theformation of these patches at the oocyte surface.

The commonly accepted hypothesis is that the spermatozoon interacts withthe membrane through a first receptor and that after transduction of atransmembrane signal, a cellular mechanism enables formation of thesepatches to which the spermatozoon attaches.

Research has been undertaken to determine the nature of the sperm ligandand the oocyte receptor involved. There is a protein in the spermatozoonmembrane called fertilin (Evans 2002). This molecule is an alpha betadimer the molecules of which belong to the ADAM protein family (ADisintegrin And Metalloprotease) (Evans 2001). The presence of integrinshas been demonstrated on the oocyte. Sperm fertilin, through itsputative binding site, can interact with one or more oocyte integrins.

Membrane molecules called integrins are binding molecules which play arole in cell-extracellular matrix and cell-cell binding. Their ligandsbind to their extracellular domain via a binding site composed of atripeptide located at the tip of a loop.

Spermatozoa contain a membrane molecule with a disintegrin site that canbind integrin. This molecule, discovered in the guinea pig but presentin all mammals studied so far including humans and mice, is calledfertilin. Human fertilin has been sequenced and so its putative bindingsite is known, which is the FEE tripeptide (phenylalanine, glutamicacid, glutamic acid) (Gupta et al. 1995; Vidaeus et al.; 1997). A linearoctapeptide containing the FEE sequence inhibits the adhesion andpenetration of sperm to the oocyte (Bronson 1999). Similar inhibitoryeffects have been observed in other species with linear and cyclicpeptides (Mwethera 1999; Gupta 2000; Li 2002; Myles 1994). Evans (1995)reported an absence of inhibitory effect of cyclic peptides in mice.

DESCRIPTION OF THE INVENTION

During their research on inhibitors of fertilization, the inventorssynthesized a cyclic peptide called FEEc containing the binding site ofhuman fertilin beta and tested the effect thereof on human oocytes. In asurprising and unexpected manner, the inventors thereby showed that (1)the cyclic peptide FEEc binds the human oocyte membrane, (2) it inducesdisplacement of adhesion proteins to the oocyte surface, saiddisplacement normally being induced by spermatozoa, (3) it increases thefusiogenic capacity of oocytes. These findings were corroborated byexperiments in mice.

Thus, the inventors have identified a novel class of cyclic peptidescapable of increasing the fusiogenic capacity of oocytes and/orspermatozoa. In addition, said class of cyclic peptides would presumablybe capable of activating oocytes.

The invention relates to the use of a cyclic peptide comprising thebinding site of fertilin beta (ADAM 2) to oocyte integrin in order inorder to increase the fusiogenic capacity and/or to active an oocyte,typically in vitro or ex vivo. Said binding site is contained in thedisintegrin loop of fertilin beta. Said cyclic peptide minimallycomprises the tripeptide essential for this binding. This tripeptidediffers according to species. However, the organization of thedisintegrin loop is very highly conserved between species (Table 1). Itwill therefore be easy for those skilled in the art to define thedisintegrin loop of fertilin beta and identify the tripeptide.

TABLE 1  Disintegrin loop Human ₍₄₃₆₎ CLFMSKERMC RPS FEE CDLP EYCNGSSASC₍₄₆₅₎ SEQ ID No 1 Mouse ₍₄₄₀₎ CKLKRKGEVC RLA QDE CDVT EYCNGTSEVC₍₄₆₉₎ SEQ ID No 2 Guinea pig ₍₄₃₃₎ CEEKTKGEVC RES TDE CDLP EYCNGSSGAC₍₄₆₂₎ SEQ ID No 3 Rabbit ₍₄₃₅₎ CTFKERGQSC RPP VGE CDLF EYCNGTSALC₍₄₆₄₎ SEQ ID No 4 Macaque ₍₄₃₆₎ CLFMSQERCC RPS FDE CDLP EYCNGTSASC₍₄₆₅₎ SEQ ID No 5 Bovine ₍₄₃₅₎ CAFIPKGHIC RGS TDE CDLH EYCNGSSAAC₍₄₆₄₎ SEQ ID No 6 Rat ₍₄₄₁₎ CNLKAKGELC RPA NQE CDVT EYCNGTSEVC₍₄₇₀₎ SEQ ID No 7 Pig ₍₄₃₅₎ CSFMAKGQTC RLT LDE CDLL EYCNGSSAAC₍₄₆₄₎ SEQ ID No 8 The positions shown in parentheses correspond to thepositions in the fertilin beta sequences referenced hereinafter.Residues underlined in bold type correspond to the tripeptide. Residuesin bold type are perfectly conserved between species

A consensus sequence for the disintegrin loop can be deduced from Table1.

(SEQ ID NO: 12) C-X₂-(F/L)-(K/M/I)-X₅-(K/R/Q)-(G/E)-X₈-X₉-C-R-X₁₂-X₁₃-TriPept-C-D-(L/V)-X₂₀-E-Y-C-N-(G/E)-(T/S)-S- (A/E/G)-X₂₉-C wherein the X groups represent, independently of one another, an aminoacid and “TriPept” is the tripeptide essential for binding of fertilinto integrin. Preferably, X₈ is a charged amino acid. More particularly,it is selected in the group consisting of E, R and Q. Preferably, X₁₂ isselected in the group consisting of P, L, E, and G. Preferably, X₁₃ is asmall and uncharged amino acid, more particularly selected in the groupconsisting of S, A, P, and T. Preferably, X₂₉ is a small and unchargedamino acid, more particularly selected in the group consisting of S, A,and V.

The cyclic peptide can be cyclized by any method known to those skilledin the art. The peptide can be cyclized by means of a covalent bondbetween the main chain and the main chain, between the main chain and aside chain, or between a side chain and another side chain. Said bondcan be a disulfide, amide or thioether bond.

For example, the peptide can be cyclized by a peptide bond between theN-terminal residue and the C-terminal residue, or with amino orcarboxylic groups of the side chains of the residues.

Preferably, the peptide is cyclized by means of two cysteine residues,more particularly by means of a disulfide bridge between said twocysteine residues. The cysteine residues must be located in such as wayas to permit cyclization of the peptide. The cysteine residues can belocated in such a way that, after cyclization, the peptide has apolypeptide tail. Preferably, said cysteine residues are located at theends of the peptide.

The cyclic peptide according to the invention can therefore be describedby the following formula:

wherein X represents an amino acid, m and n are comprised between 0 and14. As indicated earlier, in the inventive formulas, the X groups areindependent of one another and can represent, within a same molecule,amino acids which are the same or different. Preferably, when m or n isequal to 0, the other is at least 1. Preferably, m+n is less than 10,preferably less than or equal to 5. In a preferred embodiment, m+n isequal to 3. Preferably, the tripeptide contains a sequence X-(Q/D/E)-E,preferably X-(D/E)-E. For example, the tripeptide can be selected in thegroup consisting of (Q-D-E), (F-E-E), (T-D-E), (V-G-E), (F-D-E),(T-D-E), (N-Q-E), (L-D-E). In a preferred embodiment, the tripeptide is(F-E-E).

In a preferred manner, the cyclic peptide according to the invention isdescribed by the following formula:

The cysteine residues involved in peptide cyclization can be naturallylocated in the disintegrin loop or can be introduced into the peptidesequence. The disintegrin loops are rich in cysteine. In fact, cysteineresidues are conserved at positions positions 1, 10, 17, 23 and 30 ofsaid loops. Thus, the peptides can be cyclized by means of a disulfidebridge selected in the group consisting of: C1-C17, C1-C23, C1-C30,C10-C17, C10-C23, and C10-C30. Preferably, the peptides are cyclized bymeans of a disulfide bridge selected from C10-C17 and C10-C23.

The cyclic peptides according to the invention can therefore display oneof the following structures:

(SEQ ID NO: 10) C₁-X2-(F/L)-(K/M/I)-X₅-(K/R/Q)-(G/E)-X₈-X₉-C-R-X₁₂-X₁₃-TriPept-C₁₇; (SEQ ID NO: 11)C₁-X2-(F/L)-(K/M/I)-X₅-(K/R/Q)-(G/E)-X₈-X₉-C-R-X₁₂-X₁₃-TriPept-C-D-(L/V)-X₂₀-E-Y-C₂₃; (SEQ ID NO: 12)C₁-X2-(F/L)-(K/M/I)-X₅-(K/R/Q)-(G/E)-X₈-X₉-C-R-X₁₂-X₁₃-TriPept-C-D-(L/V)-X₂₀-E-Y-C-N-(G/E)-(T/S)- S-(A/E/G)-X₂₉-C₃₀;(SEQ ID NO: 13) C₁₀-R-X₁₂-X₁₃-TriPept-C₁₇; (SEQ ID NO: 14)C₁₀-R-X₁₂-X₁₃-TriPept-C-D-(L/V)-X₂₀-E-Y-C₂₃; (SEQ ID NO: 15)C₁₀-R-X₁₂-X₁₃-TriPept-C-D-(L/V)-X₂₀-E-Y-C-N-(G/E)-(T/S)-S-(A/E/G)-X₂₉-C₃₀;wherein X represents an amino acid and the cysteine residues at the endsof the peptide form disulfide bridges.

Preferably, the cyclic peptides according to the invention display oneof the following structures:

(SEQ ID NO: 13) C₁₀-R-X₁₂-X₁₃-TriPept-C₁₇; or (SEQ ID NO: 14)C₁₀-R-X₁₂-X₁₃-TriPept-C-D-(L/V)-X₂₀-E-Y-C₂₃.

More particularly, the cyclic peptides according to the invention areselected in the group consisting of the fragments 1-17, 1-23, 1-30,10-17, 10-23, and 10-30 of one of the sequences SEQ ID Nos 1-8,preferably of sequence SEQ ID No 1. Preferably, the cyclic peptidesaccording to the invention are selected in a group consisting offragments 10-17, 10-23 of one of the sequences SEQ ID Nos 1-8,preferably of sequence SEQ ID No 1.

The cysteine residues can be introduced into the peptide to be cyclized.In a preferred embodiment, the inventive peptide has the followingstructure:

wherein X is an amino acid. A small and uncharged amino acid will bepreferred.

Preferably, X is selected in the group consisting of A, S or T. Moreparticularly, X is A or S. In a preferred embodiment, X is serine andthe tripeptide has the sequence (F-E-E) (SEQ ID No 9). Moreparticularly, the invention relates to said cyclic peptide (named FEEcin the examples) and to the use thereof for increasing the fusiogeniccapacity of and/or for activating oocytes.

The amino acids of the cyclic peptide according to the invention can benatural or not. A non-natural amino acid denotes an analogue orderivative of a natural amino acid. For example, a non-natural aminoacid can have a longer, shorter or different side chain containingsuitable functional groups. It is understood that consideration is takenof L and D stereoisomers. In addition, the peptide bonds can be modifiedto make them resistant to proteolysis. For example, at least one(—CO—NH—) peptide bond can be replaced by a divalent bond selected inthe group consisting of (—CH₂—NH—), (—NH—CO—), (—CH₂—O—), (—CH₂—S—),(—CH₂—CH₂—), (—CO—CH₂—), (—CHOH—CH₂—), (—N═N—), and (—CH═CH—).

The residues of the sequences described hereinabove can vary in aconservative manner. Conservative is understood to mean that the variantresidue displays similar physico-chemical characteristics. Sterichindrance, polarity, hydrophobicity or charge are among thephysico-chemical characteristics taken into account.

Consequently, the invention also relates to variants and/or derivativesof said cyclic peptides and to the use thereof, particularly in order toincrease the fusiogenic capacity and/or to activate oocytes. Saidvariants and derivatives conserve the binding capacity to oocyteintegrin, more particularly of integrin α6β1.

The invention also relates to a multimer of the inventive cyclicpeptide. This polymerization of the cyclic peptide can be achieved byany method known to those skilled in the art. Preferably, the cyclicpeptide is coupled with a carrier molecule allowing the peptide topolymerize. The bond between the cyclic peptide and the carrier moleculecan be covalent or noncovalent. The methods by which to attach thecyclic peptide to the carrier molecule are well known to those skilledin the art and comprise amine chemistry, carbodiimide coupling ofcarboxyl and amino derivatives, activation of cyanogen bromide,N-hydroxysuccinimide, epoxide, sulfhydryl, or hydrazide. The bondbetween the carrier molecule and the cyclic peptides can be direct orindirect. When it is indirect, it can take place through a linker. Saidlinker can play a role of spacer which avoids interference of thecarrier molecule on the properties of the cyclic peptide, particularlythe fusiogenic and/or activator properties. Said linker can be apeptide. The cyclic peptide must be attached to the carrier molecule insuch a way as to maintain the accessibility of the tripeptide. Thenumber of cyclic peptides comprised in the multimer is preferablycomprised between 2 and 1000. The multimeric form of the inventivepeptide makes it possible to increase the effect of the cyclic peptideon the displacement of oocyte adhesion proteins.

For example, the polymerization can be accomplished by means of abiotin/streptavidin conjugate which allows a tetramer of the cyclicpeptide to be prepared, whereby each cyclic peptide is bound to a biotinand four biotins can bind to a streptavidin molecule.

Alternatively, several cyclic peptides according to the invention can becarried on a transport protein. Preferably, said protein does not haveany biological effect on the oocyte. Those skilled in the art know ofseveral proteins used as carrier proteins including bovine serum albumin(BSA) and limpet hemocyanin.

Moreover, several cyclic peptides according to the invention can beimmobilized on a solid support. Non-limiting examples of the solidsupport are agarose, glass, cellulose resins, silica resins,polystyrene, and polyacrylamide. The solid support can be modified withfunctional groups allowing fixation of the cyclic peptides, for exampleby means of carboxyl, amino, sulfhydryl, hydroxyl and/or carbohydrategroups contained in said peptides.

The invention concerns a composition comprising a cyclic peptideaccording to the invention or a multimer of the same. Said compositioncan be a medium intended for culturing gametes. Said composition canalso comprise a pharmaceutically acceptable support. Said compositioncan be a pharmaceutical composition, preferably suitable for localapplication. Preferably, said local application takes place in thefemale genital tract.

The invention concerns the use of a cyclic peptide according to theinvention, a multimer of the same or a composition according to theinvention in order to increase the fusiogenic capacity of a gamete, inparticular the oocyte, or to activate an oocyte, and a correspondingmethod.

In particular, the cyclic peptides according to the invention can beused for in vitro fertilization, for artificial insemination and fornuclear transfer (cloning) in mammals, preferably non-human. Preferably,said peptides will be used to carry out or to improve in vitrofertilization, more particularly in humans. In this respect, theinvention also concerns a gamete culture medium supplemented with acyclic peptide according to the invention or a multimer of the same. Ina particular embodiment, the oocyte is contacting with the cyclicpeptide according to the invention before being incubated with sperm. Inanother embodiment, the oocyte is contacting simultaneously with thecyclic peptide according to the invention and the male gamete.

The inventive peptides can also be used in a treatment designed toenhance fertility. Thus the invention concerns the use of an inventivepeptide for preparing a medicament intended for the treatment offertility problems, and a corresponding method.

The cyclic peptides according to the invention can also be used topotentiate spermatozoa.

The different methods and uses of the invention can be carried out invitro, ex vivo or in vivo. Advantageously, they are carried out in vitroor ex vivo so as to produce treated biological materials or cells.

The cyclic peptides according to the invention can be used for anyspecies the reproduction of which involves gametes. The invention is ofparticular interest for endangered species, species with low fertilityor highly valuable species. More particularly, the inventioncontemplates the use of the cyclic peptides in mammals. Preferably, theinvention can be used in the reproduction or cloning of ovines, bovines,equines, etc. It is understood that the invention can also be used toassist human procreation.

Said peptide added to culture media for human gametes can potentiate thefusiogenic capacity of the latter and lead to the formation of embryosall while not interfering with physiological membrane interactions. Theuse of said peptide has two potential interests: reducing the use ofintracytoplasmic microinjection as a fertilization technique andimproving the quality of the embryos obtained.

The invention further concerns methods for screening compoundsincreasing the fusiogenic capacities of the oocyte. Said methodcomprises the following steps: 1) incubating the oocyte in the presenceof the test compound; and 2) evaluating the ability of the compound toincrease the fusiogenic capacity of the oocyte. In a preferred manner,fusiogenic capacity can be estimated by at least one of the followingcriteria: binding to the oocyte, induction of adhesion proteindisplacement to the oocyte surface (more particularly of integrin α6β1),fusion of the oocyte with a spermatozoon. Preferably, displacement ofadhesion proteins to the oocyte surface leads to the formation ofpatches of adhesion proteins (more particularly of integrin α6β1). Thedisplacement of adhesion proteins can be evaluated by the labelling ofthe same.

The invention relates to compounds inducing the redistribution of oocyteadhesion proteins into patches and to the use thereof for increasing thefusiogenic capacities of oocytes.

Other aspects and advantages of the invention will become apparent inthe following examples, which are given for purposes of illustration andnot by way of limitation.

EXAMPLES Results of the Study of Cyclic Peptide FEEc on Human GameteFusion Materials and Methods

Human Oocytes

The human oocytes used in the experiments were from two sources: oocyteswhich failed to be fertilized after IVF and oocytes which were notmicroinjected due to immaturity at the time of ICSI and matured invitro. Patients donated said oocytes for research purposes and signed aconsent form approved by the Institutional Review Board of theAulnay-sous-Bois hospital.

These oocytes aged 48 hours or matured in vitro were not or were nolonger capable of producing an embryo. Moreover, the polyspermy blockingmechanism which ensures that the embryo will be diploid is located inthe zona pellucida of the oocyte. By removing the zona pellucida ofthese oocytes, this protective mechanism is abolished, leading tononviable polyspermic zygotes in the event of gamete fusion. Finally, asembryo development occurs inside the zona pellucida for the first sixdays, the absence of the latter precludes said development from takingplace.

To maintain the oocyte membrane as close as possible to its naturalstate, the inventors removed the zona pellucida mechanically withmicrosurgical scissors.

Spermatozoa

Sperm from fertile donors was collected after three days of abstinenceand prepared as for IVF. Briefly, the ejaculate was placed at 37° C.until liquefication and then selected on a 2 layer Puresperm gradient(90 and 45%). The sperm was then kept in capacitating conditions untilinsemination of the oocytes.

Synthetic Cyclic Peptide FEEc

The complete sequence of the disintegrin domain of human fertilinconstituting the loop is shown below:

SEQ ID No 1 CLFMSKERMCRP SFEE CDLPEYCNGSSASC

The synthetic cyclic peptide FEEc used in this example is as follows:

SEQ ID No 9 C SFEEC

The FEEc peptide synthesized by the inventors comprises the sequence ofthe FEE tripeptide. It comprises the Serine preceding said sequence andthe Cysteine following it. The inventors introduced another Cysteineupstream of the binding site. The two cysteines located at the ends ofthe peptide enabled the cyclization thereof.

Immunofluorescence

The integrin alpha 6 subunit is present on the human oocyte membrane. Itforms part of the multimolecular fusion complex. It is for this reasonthat the inventors used it as a control of membrane reorganization.

Zona-free oocytes were incubated in 20 μl drops of Ferticult culturemedium under oil at room temperature. The medium was supplemented with20 μM mouse anti-human alpha 6 antibody (Chemicon International, London,GB). The oocytes were then washed and fixed in 2% PFA for 1 hour. Theywere then incubated for 45 minutes with a second donkey anti-mouseimmunoglobulin antibody labelled with FITC or rhodamine (FITC-conjugateddonkey anti-mouse IgG or Rhodamine-conjugated donkey anti-mouse IgG 10μg/ml; Jackson Laboratories). The oocytes were washed and mounted inantifade Immunomount (Shandon Laboratories) between the coverslip andslide and examined either under a fluorescence microscope (ZeissAxiophot) or a confocal microscope (Leica Lasertechnik, GmbH).

Functional Fusion Assays

Functional tests of inhibition of fusion were carried out in conditionssimilar to those used for IVF, that is, in conditions sufficiently closeto physiological conditions since they enable pregnancy and fetuses.Briefly, the oocytes were incubated in 20 μl drops of Ferticult underoil for 18 hours in a 5% CO2 incubator at 37° C. with 4000 mobile spermcells. The tests were carried out by supplementing the medium with theFEEc peptide at 200 μM concentration. At the end of incubation, theoocytes were washed and incubated for 20 minutes in Hoechst 33342(Sigma) 5 μg/ml. After fixation in 4% PFA 1% PBS-BSA for 30 minutes atroom temperature, the oocytes were mounted in Immunomount between thecoverslip and the slide and examined under ultraviolet light. Fusedspermatozoa were fluorescent. The slides were analyzed under a ZeissAxiophot microscope equipped with a camera connected to Imaging SystemPackage image analysis software (Applied Imaging, Newcastle-upon-Tyne,UK).

Results

Immunofluorescence and Confocal Microscopy.

The oocytes were incubated with 200 μM biotinylated FEEc peptide for 45minutes. Confocal microscopy was used to reveal labelling of themembrane with the FEEc peptide as seen on the oocyte equatorial segment(FIG. 1A). Computerized superimposition showed that this labellingcorresponded to membrane patches (FIG. 1B).

Induction of Fusion Patches by the FEEc Peptide on Human Oocytes

The spermatozoa induced the formation of multimolecular patches. Byincubating oocytes matured in vitro with a suspension of 200 μM of FEEcpeptide for 18 hours, the inventors demonstrated a redistribution of theintegrin α6 subunit. In fact, while the distribution of the α6 subuniton the surface of intact oocytes was homogeneous (FIG. 2A), the FEEcpeptide induced the redistribution thereof in the form of small membranepatches. In mice, spermatozoa induce said patches during fertilization.It can be concluded that the FEEc peptide induces a redistribution ofadhesion proteins on the membrane similar to that induced by the spermcell itself at fertilization.

Functional Fusion Assay with Human Gametes

In similar conditions, zona-free oocytes were incubated with 200 μM FEEcpeptide and spermatozoa. In the control oocytes, about twenty fusedsperm cells could be seen in the oocyte cytoplasm (FIG. 3A). In thepresence of 200 μM FEEc peptide, the number of spermatozoa in thecytoplasm was much higher (FIG. 3B). The experiment was conducted on alarger number of oocytes and revealed a mean 60% increase in the numberof spermatozoa that fused with the oocyte (26.1±8.3 vs 16.4±5.2;P<0.001). The increase in the number of spermatozoa was thereforestatistically significant. This effect was specific because incubationwith the same scrambled peptide had no effect. The action of the FEEcpeptide was reversible because oocytes preincubated with the FEEcpeptide then washed and inseminated showed no change in their fusiogeniccapacity. Thus there is no toxic effect on oocytes, althoughco-incubation is necessary for the effect of the peptide to appear.

Discussion

The FEEc peptide therefore has the property of increasing the fusiogeniccapacity of human oocytes. It mimics the mechanism by which thespermatozoon induces molecular fusion complexes on the oocyte uponcontact with the oocyte membrane and therefore facilitates the fusionprocess.

It can be used to supplement the culture media used to carry out invitro fertilization, in which case it should improve the fertilizationrate of IVF carried out for idiopathic infertility or sperm deficiency.This would lower the need for microinjection techniques and improveembryo quality.

Results of the Study of Cyclic QDE on Gamete Fusion in Mice

In line with what was observed in humans, the effect of a cyclictripeptide was studied in the mouse model. By analogy, the tripeptidemotif in this case contained the disintegrin binding site of mousefertilin 13, that is, the QDE tripeptide. This peptide was named QDEc.

Materials and Methods

Six-week-old C56b1/CBA mice were hyperstimulated with 5 IU PMSG and 5 IUhCG administered at a 48 hour interval. Thirteen hours after the lastinjection, the mice were sacrificed and the oviducts removed andlacerated in M2 medium. The oocytes were retrieved and eitherinseminated in their cumulus or decoronized by a short treatment withhyaluronidase. Decoronized oocytes were then mechanically stripped oftheir zona pellucida with microdissection scissors under a binocularmagnifier.

Zona-free oocytes were preincubated for 30 minutes and then inseminatedin the presence of (1) M16 medium for the control group; (2) mediumsupplemented with 10 μM; (3) 100 μM QDEc and (4) 1 mM QDEc. In all casesthe oocytes were inseminated for 3 hours with 10⁶ mobile sperm per ml.

Intact ovocytes were incubated with 100 μM QDEc and 10⁵ sperm/ml.

After incubation, the oocytes were thoroughly washed and incubated in 10μM Hoescht for 30 minutes, rinsed and then examined in ultraviolightlight under a Zeiss microscope. The data were analyzed with Statview®software.

Results

1—Zona-Free Oocytes

In five experiments, 170 oocytes were studied in four groups. While themean number of fused sperm per oocyte was 2.2±0.1 (mean±SD) in thecontrol group, it increased in a dose-dependent manner when QDE waspresent in the incubation medium (FIG. 5), showing a statisticaldifference at 100 μM concentration (P<0.05) and statistical significanceat 1 mM (p<0.004), with a 32% increase in the number of fusedspermatozoa.

2—Intact Oocytes

By conventional IVF: The three experiments gave similar results with amean fertilization rate of 32.1+7.6% for the controls and 56.3+16.0% forthe test group (P<0.001) (FIG. 6).

Discussion

The number of spermatozoa that fused with a zona-free oocyte wasapproximately twenty in humans whereas in mice it generally did notexceed two. This number, which increased to a lesser extent than inhumans under the effect of QDE, rapidly reached statisticalsignificance. Furthermore, during in vitro fertilization of intactoocytes, QDEc led to a 75% increase in the normal oocyte fertilizationrate. The interaction of the spermatozoon with the oocyte membranetherefore appears to be similar in mice and humans. The cyclized versionof the peptide which mimics the disintegrin binding site of fertilinbeta clearly increases the fusiogenic capacity of gametes.

LEGENDS OF FIGURES

FIG. 1. Detection of biotinylated FEEc peptide at the surface of humanoocytes.

The zona pellucida was mechanically removed from the oocytes which werethen incubated for 45 minutes with 100 μM biotinylated peptide. The FEEcpeptide was detected with a mouse anti-biotin antibody recognized by abiotinylated anti-mouse immunoglobulin antibody then bystreptavidin-FITC. The oocytes were then mounted in immunomount betweenthe coverslip and the slide and examined under a confocal microscope, B:superimposition of sections corresponding to a hemi-oocyte.

FIG. 2. Induction of fusion patches on human oocytes by FEEc peptide.

Intact oocytes were incubated with 200 μM FEEc peptide for 45 minutes.They were washed, incubated with 20 μM anti-integrin α6 subunit antibodyfor 45 minutes, then fixed in 4% PFA for 30 minutes. They were washed,incubated with a second rhodamine-labelled anti-mouse immunoglobulinantibody and examined by immunofluorescence. FIG. 2A: control oocyteshowing diffuse, homogeneous fluorescence on the oocyte surface. FIG.2B: after incubation with FEEc peptide, the α6 subunit redistributed toform membrane patches.

FIG. 3. Functional fusion assay with human gametes.

Zona-free oocytes were incubated in 20 μl drops of culture medium underoil with 4000 mobile spermatozoa for 18 hours. They were then washed andincubated in 10 μM Hoechst 33342 for 30 minutes and washed again,mounted in immunomount between the coverslip and the slide and examinedunder ultraviolet light. FIG. 3A: control oocyte showing the presence ofabout twenty fused spermatozoa in the oocyte cytoplasm. FIG. 3B: oocyteco-incubated with 100 μM FEEc peptide showing about sixty fusedspermatozoa.

FIG. 4. Effects of the FEEc peptide on fertilization of human oocytes.

Comparison of fertilization rates obtained in the presence and absenceof 100 μM FEEc peptide in the incubation medium.

FIG. 5. Effects of the FEEc peptide on gamete fusion in mice.

The numbers in parentheses represent the number of oocytes in eachgroup. Statistically different from control group: * (P<0.05); **(P<0.04)

FIG. 6. Effect of QDEc on in vitro fertilization of intact oocytes inmice.

REFERENCES

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We claim:
 1. A method for increasing fusiogenic capacities of a gametecomprising contacting the gamete with a cyclic peptide consisting of thefollowing formula:

wherein X represents an amino acid which can be different or identicalfor each occurrence, and m+n is less than or equal to
 5. 2. The methodaccording to claim 1, wherein the cyclic peptide consists of thefollowing formula:

(SEQ ID NO: 17) where X is an amino acid.
 3. The method according toclaim 2, wherein X is selected from the group consisting of A, S and T.4. The method according to claim 3, wherein X is S.
 5. The methodaccording to claim 1, wherein the gamete is an oocyte.
 6. The methodaccording to claim 2, wherein the gamete is an oocyte.
 7. A method forincreasing fusiogenic capacities of a gamete comprising contacting thegamete with a multimer of cyclic peptides, said peptides consisting ofunits of the following formula:

wherein X represents an amino acid which can be different or identicalfor each occurrence, and m+n is less than or equal to
 5. 8. The methodaccording to claim 7, wherein the cyclic peptides consist of thefollowing formula:

(SEQ ID NO:17) where X is an amino acid.
 9. The method according toclaim 8, wherein X is selected form the group consisting of A, S and T.10. The method according to claim 9, wherein X is S.
 11. The methodaccording to claim 7, wherein the gamete is an oocyte.
 12. A method forincreasing fusiogenic capacities of a gamete, comprising administeringin a female genital tract a cyclic peptide consisting of the followingformula:

wherein X represents an amino acid which can be different or identicalfor each occurrence, and m+n is less than or equal to
 5. 13. The methodaccording to claim 12, wherein the cyclic peptide consists of thefollowing formula:

(SEQ ID NO:17) where X is an amino acid.
 14. The method according toclaim 13, wherein X is selected from the group consisting of A, S and T.15. The method according to claim 14, wherein X is S.